CN113699109B

CN113699109B - Construction method of in-vitro modularized neuron network

Info

Publication number: CN113699109B
Application number: CN202111015060.7A
Authority: CN
Inventors: 石青; 陈喆; 孙韬; 王化平; 魏子厚; 陈勰
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2023-08-11
Anticipated expiration: 2041-08-31
Also published as: CN113699109A

Abstract

The invention relates to a construction method of an in-vitro modularized neuron network, which is based on a digital image processing technology, generates patterns covering all structural parameter combinations to be researched, and splices the patterns into a single pattern, and combines a soft lithography process based on laser direct writing and a surface modification method to prepare a micro printing plate containing all patterns to be researched so as to realize single printing, namely to generate modularized protein patterns containing any different parameter combinations, and realize in-vitro construction of the modularized neuron network composed of real neurons and different parameter combinations.

Description

Construction method of in-vitro modularized neuron network

Technical Field

The invention relates to the technical field of tissue engineering, in particular to a construction method of an in-vitro modularized neuron network.

Background

Numerous studies have shown that in the brains of many animals, including humans, neurons are not uniformly distributed to form an integral neural network, but rather are formed in a modular, distributed structure to form individual, localized, and extremely dense, integrated units of neurons, with relatively sparse axon projections connected to synapses between units. Therefore, the research of the modularized neuron network plays a very important role in researching the memory and learning behaviors of the higher animals and further understanding the consciousness generation of human beings. However, the research on the modularized neuron network is focused on the research on nerve circuits formed by specific brain regions in living rodents, and the circuits have important research value because the circuits themselves carry important sensory or motor functions and inherently have nerve coding and decoding functions. However, also because of its complete codec function, many studies can only link the behavior level with the discharge level of a part of neurons in an attempt to understand its codec. Such studies are limited by the inability to change or customize important parameters of the modular neuronal network, and thus the progress of the research in terms of the codec mechanism of the modular neuronal network is slow. Therefore, the in vitro construction of the modularized neuron network composed of different parameter combinations of real neurons is very important for researching the encoding and decoding modes of the modularized neuron network, researching the influence of information transmission between structural parameters and the network and the dynamic characteristics of the modularized neuron network, and researching the interrelation between the parameters and pharmacological actions.

Disclosure of Invention

The invention aims to provide a construction method of an in-vitro modularized neuron network, which is used for realizing in-vitro construction of the modularized neuron network composed of real neurons and different parameter combinations.

In order to achieve the above object, the present invention provides the following solutions:

a method of constructing an in vitro modular neuronal network, the method comprising the steps of:

generating mask patterns covering all structural parameters to be researched by adopting a digital image processing method;

preparing a PDMS micro-printing plate according to the mask pattern by adopting a laser direct writing soft lithography process;

printing a protein pattern in the surface-modified culture dish according to the PDMS micro-printing plate to obtain the culture dish printed with the protein pattern;

and (3) inoculating primary cortical neurons on the culture dish printed with the protein patterns to obtain an in-vitro modularized neuron network.

Optionally, the generating a mask pattern covering all structural parameters to be studied by using a digital image processing method specifically includes:

programming a modularized pattern with adjustable drawing parameters;

splicing the modularized patterns with adjustable parameters to obtain spliced patterns;

and adjusting the parameters of each modularized pattern with adjustable parameters in the spliced patterns to generate a mask pattern covering all structural parameters to be researched.

Optionally, programming the modular pattern with adjustable drawing parameters specifically includes:

generating a product containing p _row ×p _col A module value matrix of individual elements; wherein p is _row And p _col Representing the number of transverse elements and longitudinal elements of the module value matrix;

resetting all elements in a square area with the upper left corner ((i-1) multiplied by 2n+1, (j-1) multiplied by 2n+1) and the lower right corner ((i-1) multiplied by 2n+n, (j-1) multiplied by 2n+n) in the module numerical matrix to 255, and finishing drawing of the module area of the modularized pattern with adjustable parameters; wherein i and j respectively traverse from 1 to N _row And N _col N is the side length of the module, N _row The number of modules, N, included in the transverse direction for a parametrically adjustable modular pattern _col The number of modules included in the longitudinal direction for the parameter-adjustable modular pattern;

the upper left corner in the module numerical matrix is ((i-1). Times.2n+round (n/(X) _con +1))×(k-1)-x _width The coordinates of the lower right corner element are ((i-1) ×2n+round (n/(X)) _con +1))×(k-1)+x _width Setting all elements in the (j-1) multiplied by 2n+3n/2) area to 255, and finishing drawing of a transverse connection band between the j-th and j+1th modules of the i-th row; wherein k traverses from 1 to X _con ，X _con For the number of transverse connecting bands between the j-th and j+1th blocks of the i-th row, round () is the rounding operator, x _width A bandwidth of a transverse connection band between the jth and the (j+1) th modules of the ith row;

the upper left corner is ((j-1) ×2n+round (n/(Y) _con +1))×(k'-1)-y _width The coordinates of the lower right corner element are ((j-1) ×2n+round (n/(Y)) _con +1))×(k'-1)+y _width 2, setting all elements in the (i-1) multiplied by 2n+3n/2) area as 255, and finishing drawing of longitudinal connection bands between the ith and the (i+1) modules of the jth column; wherein k' traverses from 1 to Y _con ，Y _con For the number of longitudinal pass bands between the ith and the (i+1) th module of the jth column, round () is the rounding operator, y _width The bandwidth of the longitudinal passband between the ith and the (i+1) th module of the jth column.

Optionally, the splicing the plurality of modularized patterns with adjustable parameters to obtain a spliced pattern specifically includes:

generating an inclusion P _row ×P _col A splice numerical matrix of individual elements;

wherein P is _row Representing the number of transverse elements, P, in the splice value matrix _col Representing the number of longitudinal elements in the data matrix,f _row and f _col Respectively representing the number of transverse arrangements and longitudinal arrangements of the parameter-adjustable modularized patterns to be spliced g _row And g _col Respectively the transverse spacing and the longitudinal spacing between the modularized patterns with adjustable parameters to be spliced; p is p _row And p _col Representing the number of transverse elements and longitudinal elements of the module value matrix;

copying the element values of the (x, y) th parameter-adjustable modularized pattern to the upper left corner of the spliced numerical matrix to be (p) _row ×(x-1)+1,p _col X (y-1) +1) and the lower right corner is (p) _row ×(x-1)+p _row ,p _col ×(y-1)+p _col ) Wherein x and y traverse from 1 to f, respectively _row And f _col 。

Optionally, the adjustable parameters in the parameter-adjustable modular pattern include: the side length of the modules, the center distance of the modules, the number of communication bands between the modules and the width of the communication bands are used for adjusting the number of neurons, and the center distance of the modules, the number of the communication bands between the modules and the width of the communication bands are used for adjusting the communication intensity between the neurons.

Optionally, the preparing the PDMS micro-printing plate according to the mask pattern by using a soft lithography process of laser direct writing specifically includes:

loading the mask pattern into a mask generator to perform laser exposure mask on the chromium plate to generate a masked chromium plate;

developing and etching the chromium plate after masking to obtain a die;

and pouring the mixed solution of the PDMS prepolymer and the curing agent into the mold to be solidified into glue, thus obtaining the PDMS micro-printing plate.

Optionally, the protein pattern is generated in the surface modified culture dish according to the PDMS micro printing plate, and the culture dish printed with the protein pattern is obtained, which specifically comprises:

dropping agarose water solution with concentration of 0.2% in a circular area with center diameter of 25mm of a 35mm polyethylene culture dish, and carrying out ultraviolet irradiation and natural air drying to obtain a surface modified culture dish;

placing the PDMS micro-printing plate with the pattern face downwards at the bottom of the surface modified culture dish;

and after the balance weight is pressed on the PDMS micro-printing plate for a preset period of time, the balance weight and the PDMS micro-printing plate are taken down, and the culture dish printed with the protein pattern is obtained.

Optionally, primary cortical neurons are inoculated on the culture dish printed with the protein pattern, and an in-vitro modularized neuron network is obtained, and the method further comprises the following steps:

placing a semi-hollow annular sleeve in the central region of a 35mm dish;

pouring a mixed solution of PDMS prepolymer and curing agent into a 35mm culture dish for solidification and gel formation to obtain a PDMS annular structure, and stripping the PDMS annular structure from the 35mm culture dish;

two through holes are drilled at 180-degree intervals in the PDMS annular structure, and two through holes are drilled at 180-degree intervals in the side wall of the culture dish printed with the protein pattern;

placing the PDMS ring structure with the through holes in the culture dish with the through holes and the protein patterns printed thereon, and respectively corresponding the two through holes of the PDMS ring structure with the positions of the two through holes of the culture dish with the protein patterns printed thereon;

one pipeline sequentially passes through one through hole of the culture dish printed with the protein pattern and one through hole of the PDMS annular structure, and the other pipeline sequentially passes through the other through hole of the culture dish printed with the protein pattern and the other through hole of the PDMS annular structure and is fixed, so that the culture dish printed with the protein pattern after the restraint of the PDMS annular structure is added is obtained.

According to the specific embodiment provided by the invention, the invention discloses the following technical effects:

the invention provides a construction method of an in-vitro modularized neuron network, which comprises the following steps: generating mask patterns covering all structural parameters to be researched by adopting a digital image processing method; preparing a PDMS micro-printing plate according to the mask pattern by adopting a laser direct writing soft lithography process; printing a protein pattern in the surface-modified culture dish according to the PDMS micro-printing plate to obtain the culture dish printed with the protein pattern; and (3) inoculating primary cortical neurons on the culture dish printed with the protein patterns to obtain an in-vitro modularized neuron network. The invention can generate patterns covering all structural parameter combinations to be researched based on a digital image processing technology, splice the patterns into a single pattern, and combine a soft lithography process based on laser direct writing and a surface modification method to prepare a micro printing plate containing all patterns to be researched so as to realize single printing, namely to generate modularized protein patterns containing any different parameter combinations, and realize in-vitro construction of modularized neuron networks composed of real neurons and different parameter combinations.

The invention also utilizes the micro-fluidic technology to carry out open type constraint on the substrate subjected to surface modification and patterning, and has the function of supporting contact electrophysiological detection on the basis of effectively improving inoculation density and having a pourable channel to support the function of accurate drug testing.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method of constructing an in vitro modularized neuronal network according to the present invention;

FIG. 2 is a schematic diagram of a method of constructing an in vitro modular neuronal network according to the present invention;

FIG. 3 is a diagram of an example of a modular pattern provided by the present invention;

FIG. 4 is a diagram of an example of a mask pattern provided by the present invention;

FIG. 5 is a flow chart of the preparation of PDMS micro-printing plate according to the present invention;

FIG. 6 is a flow chart of a printing generated protein pattern provided by the present invention;

FIG. 7 is a flow chart of adding PDMS ring structure constraints provided by the present invention;

fig. 8 is a schematic structural diagram of a culture dish printed with a protein pattern after adding PDMS annular structure constraint provided in the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.

The invention combines the digital image generation and processing technology, the micro-contact printing technology, the numerical control processing technology and the micro-flow control technology, has the advantages of high throughput, adjustable inoculation density, high controllability, high expansibility of supporting drug test and contact electrophysiology measurement by perfusion and the like, can cover all network structure parameters by single printing, and can be used for forming a reliable modularized network with multiple structure parameters, wherein the coverage parameters comprise the number and density of neurons in modules, the communication intensity among modules and the like. In addition, the network also comprises a perfusion channel and an open design, can simultaneously support the functions of a contact electrophysiological measuring means such as patch clamp and the like, and the application of neurotransmitters or synaptic antagonists and the like, and can be used as an in vitro test model of a cerebral nerve circuit.

As shown in fig. 1 and 2, as a preferred embodiment, the following embodiments of the present invention are provided:

step 101, a mask pattern covering all structural parameters to be studied is generated by adopting a digital image processing method.

Step 101, generating a mask pattern covering all structural parameters to be studied by using a digital image processing method, which specifically includes: programming a modularized pattern with adjustable drawing parameters; splicing the modularized patterns with adjustable parameters to obtain spliced patterns; and adjusting the parameters of each modularized pattern with adjustable parameters in the spliced patterns to generate a mask pattern covering all structural parameters to be researched.

Further, step 101 specifically includes:

programming a writer draws a modular pattern. The modularized pattern is composed of a module area and a communication area, wherein the module area is a square area of (n multiplied by n) pixels 2, and the module center distance is n _width The communication area is B _num Root bandwidth of x _width A band-shaped region of pixels. The programming is divided into two major steps: generating F from design parameters _num Images with different parameters; all images were fused into one image to produce protein patterns of all parameters in a single print.

Wherein, in the first step, the connected area is considered as B _num The pattern-free interval between the modules is also N pixels, and the pattern comprises N in transverse direction _row A module comprising N in longitudinal direction _col And a module. First, p is generated as the transverse direction and the longitudinal direction _row And p _col An element, a uint8 (unsigned 8-bit integer) matrix having an element value of 0, wherein,

next, a module region is drawn. Nesting uses two round-robin functions to reset all elements in a square region with the upper left corner ((i-1) x 2n+1, (j-1) x 2n+1) and the lower right corner ((i-1) x 2n+n, (j-1) x 2n+n) to 255, where i and j traverse from 1 to N, respectively _row And N _col 。

Again, connected band-shaped regions are drawn. Two circulation functions are adopted respectively, and the transverse and longitudinal bands are drawn in sequence. Taking a transverse band as an example, consider the bandwidth as x _width X between the ith row, the jth and the (j+1) th module _con The coordinates of the upper left and lower right corner elements of the root passband are ((i-1) ×2n+round (n/(X) _con +1))×(k-1)-x _width (j-1) ×2n+n/2) and ((i-1) ×2n+round (n/(X) _con +1))×(k-1)+x _width 2, (j-1) X2n+3n/2), where k traverses from 1 to X _con Round () is a rounding operator, setting all elements of the above region to 255. The modular pattern obtained through the above steps is shown in fig. 3.

And secondly, on the basis of obtaining modularized patterns with different parameters, all the patterns are spliced into one pattern, so that single printing can be realized to generate all the protein patterns, and the splicing realization flow is as follows. Consider a common f _n A pattern to be spliced f _n ＝F _num The layout of the total pattern is f _row ×f _col I.e. transverse and longitudinal respectively comprise f _row And f _col A sheet pattern. Wherein the method comprises the steps of

Then, first generate a containing P _row ×P _col A matrix of ui 8 values with element values of 0, wherein,

g _row and g _col Respectively, the sub-picture spacing in the transverse and longitudinal directions.

Second, nesting uses two loopsA loop function of f _x The element values of the images to be spliced are copied to the upper left and lower right element coordinates of (p) _row ×(i-1)+1,p _col X (j-1) +1) and (p _row ×(i-1)+p _row ,p _col ×(j-1)+p _col ) Wherein x= (1, 2, …, n), i and j traverse from 1 to f, respectively _row And f _col ，x＝f _col X (i-1) +j. Finally, the matrix is stored as a bitmap in the bmp format.

So far, only the parameter value needs to be changed, the modularized network generating any parameter can be covered, and the adjustable parameters of the method comprise the following steps: width n of square module region, module center-to-center spacing n _width Number of communication bands B of the communication region _num Width x _width The width n is used for adjusting the quantity of neurons of the modularized network, and the rest parameters are used for adjusting the communication intensity among the modules. The mask pattern obtained through the above steps is shown in fig. 4.

And 102, preparing the PDMS micro-printing plate according to the mask pattern by adopting a laser direct writing soft lithography process.

Step 102, preparing a PDMS micro-printing plate according to the mask pattern by using a soft lithography process of laser direct writing, which specifically includes: loading the mask pattern into a mask generator to perform laser exposure mask on the chromium plate to generate a masked chromium plate; developing and etching the chromium plate after masking to obtain a die; and pouring the mixed solution of the PDMS prepolymer and the curing agent into the mold to be solidified into glue, thus obtaining the PDMS micro-printing plate.

Further, as shown in fig. 5, step 102 specifically includes:

2-1) A spin chrome plate coated with a 12 μm thick paste was purchased, split into small chrome plates, and the small chrome plates of the appropriate size were placed on a vacuum clamping platform of a mask generator. Firstly, changing a laser write head into a 2mm write head with highest precision, wherein a single pixel corresponds to a 200nm line width, opening software Exposure Wizard matched with a mask generator, changing an operation mode into a gray mode, exposing through a bitmap, loading a mask pattern, then determining optimal laser parameters including a laser duty ratio, laser intensity, an energy mode and the like, focusing a laser by adopting a pneumatic and optical hybrid automatic focusing mode, and finally starting the mask generator;

2-2) after the exposure is finished, closing the vacuum clamping platform, taking out the chromium plate, placing the chromium plate into a 60mm polypropylene culture dish, adding 1:4 AZ400K aqueous solution, developing for 6 minutes, and always keeping the culture dish shaking during the development period so as to avoid local sediment formation and influence on the uniformity of the development process. Washing with deionized water for three times after development is finished, and drying;

2-3) placing the developed and blow-dried chromium plate into a new culture dish, adding an etchant, etching for 45-60 seconds, keeping shaking the culture dish in the etching process, flushing with deionized water for three times after the etching is finished, and blow-drying;

2-4) placing the etched chrome plate on the object stage of the optical microscope, selecting an objective lens with proper magnification, and checking the local detail of the template pattern. If the pattern edge is clear and the structure is complete, the pattern can be used as a template of the micro printing plate, otherwise, the process is repeated until the ideal effect is obtained.

2-5) mixing the PDMS prepolymer and the curing agent according to a proper proportion, fully stirring, and placing the mixture in a low-temperature environment in a dark place to remove bubbles. A60 mm polypropylene culture dish is used as a vessel, double-layer tinfoil paper is cut down, and the culture dish is attached and used for containing a template and PDMS mixed solution. And (3) putting the template with the right side upwards on a culture dish attached with double-layer tinfoil paper, adding the PDMS mixed solution with bubbles removed, putting the whole culture dish into a constant temperature incubator at 65 ℃ for two hours, taking out the culture dish, cutting off a solidified PDMS micro-printing plate, storing the culture dish in deionized water, and taking out the culture dish before use.

After the mask is generated by the mask generator in step 102, the mask pattern is not required to be transferred to the silicon wafer, and is directly used as a mould, PDMS is poured and solidified into glue, so that the process is simpler, more convenient and direct than the traditional soft lithography process.

And step 103, printing and generating a protein pattern in the surface-modified culture dish according to the PDMS micro-printing plate to obtain the culture dish printed with the protein pattern.

Step 103, generating a protein pattern in the surface-modified culture dish according to the PDMS micro-printing plate printing, and obtaining the culture dish printed with the protein pattern, which specifically comprises the following steps: dropping agarose water solution with concentration of 0.2% in a circular area with center diameter of 25mm of a 35mm polyethylene culture dish, and carrying out ultraviolet irradiation and natural air drying to obtain a surface modified culture dish; placing the PDMS micro-printing plate with the pattern face downwards at the bottom of the surface modified culture dish; and after the balance weight is pressed on the PDMS micro-printing plate for a preset period of time, the balance weight and the PDMS micro-printing plate are taken down, and the culture dish printed with the protein pattern is obtained.

Further, as shown in fig. 6, step 103 specifically includes:

3-1) an aqueous agarose solution was prepared at a concentration of 0.2%. Firstly pouring deionized water into a beaker, placing the beaker on a magnetic stirrer, adding a magnetic stirrer, starting a hot plate heating and stirring switch, and pouring out the whole deionized water after the deionized water is boiled. This operation was repeated three times to ensure a beaker with high cleanliness. Next, 50mL deionized water and a magnetic stirrer were added, after boiling of the water, 100mg agarose powder was added, after about ten minutes, the agarose was dissolved in the water and the solution was seen to be clear. A0.15 mL agarose aqueous solution is taken by a Pasteur dropper, and uniformly covered in a circular area with the center diameter of 25mm of a 35mm polyethylene culture dish, so that the dish wall is prevented from being contacted to prevent a large amount of solution from being adsorbed by the dish wall, and the agarose concentration at the bottom of the dish is too low. Standing in a fume hood, irradiating with ultraviolet light overnight, naturally air drying, covering with a dish cover, and keeping in dry environment.

3-2) preparing a protein solution. The protein solution contained ECM gel and Poly-D-lysine, and the solvent was PBS solution.

3-3) before microcontact printing, the PDMS micro-printing plate is taken out of deionized water, put into a 50mL centrifuge tube containing 10% SDS (sodium dodecyl sulfate) aqueous solution, ultrasonically cleaned for five minutes, and then stood for ten minutes, so that a layer of SDS film is uniformly plated on the micro-printing plate, and the film can improve the separation efficiency of protein. The micro printing plate is taken out of the SDS solution, washed by deionized water and dried, the micro printing plate is placed at the bottom of a new culture dish, 200 mu L of protein solution is dripped into a pattern area, a dish cover is covered, the culture dish is transferred to a 37 ℃ incubator, and the culture dish is placed for 20 minutes. After 20 minutes, the microprinting plate was removed, rinsed with deionized water, and blow dried.

3-4) simple protein printing based on centrifuge tube weighting. The microprinting plate was turned upside down with the pattern down using tweezers and placed on the bottom of a 35mm dish modified with agarose surface. The balance weight was prepared as a 50mL centrifuge tube, 3 50g weights and the balance water were added to the inside of the centrifuge tube, and the total weight was 200g. And finally, screwing the centrifuge tube, inverting the centrifuge tube above the micro printing plate, taking down the centrifuge tube after 2 minutes, and tearing off the micro printing plate by using tweezers. And standing the printed culture dish in a fume hood, covering a dish cover after drying, wrapping by using double-layer tinfoil paper, storing in a drying environment, and avoiding ultraviolet irradiation.

In step 103, the surface of the culture dish is modified before printing so that the surface becomes unsuitable for cell attachment. After such printing, only the pattern areas printed with the proteins are suitable for cell attachment.

Step 104, primary cortical neurons are inoculated on the culture dish printed with the protein pattern, and an in-vitro modularized neuron network is obtained.

Step 104, performing primary cortical neuron inoculation on the culture dish printed with the protein pattern to obtain an in-vitro modularized neuron network, and further comprising the following steps: placing a semi-hollow annular sleeve in the central region of a 35mm dish; pouring a mixed solution of PDMS prepolymer and curing agent into a 35mm culture dish for solidification and gel formation to obtain a PDMS annular structure, and stripping the PDMS annular structure from the 35mm culture dish; two through holes are drilled at 180-degree intervals in the PDMS annular structure, and two through holes are drilled at 180-degree intervals in the side wall of the culture dish printed with the protein pattern; placing the PDMS ring structure with the through holes in the culture dish with the through holes and the protein patterns printed thereon, and respectively corresponding the two through holes of the PDMS ring structure with the positions of the two through holes of the culture dish with the protein patterns printed thereon; one pipeline sequentially passes through one through hole of the culture dish printed with the protein pattern and one through hole of the PDMS annular structure, and the other pipeline sequentially passes through the other through hole of the culture dish printed with the protein pattern and the other through hole of the PDMS annular structure and is fixed, so that the culture dish printed with the protein pattern after the restraint of the PDMS annular structure is added is obtained.

Further, as shown in fig. 7, the specific steps of adding PDMS ring structure constraints include:

4-1) machining the semi-hollow annular sleeve based on a numerical control machine tool. The method adopts the commercial cortical neurons purchased and has limited quantity, so that in order to improve the neuron inoculation density and the experimental times of single-time purchased neurons, the structure constraint is required to be carried out on a culture dish with the thickness of 35mm, and the area of the non-patterned area is reduced as much as possible. Firstly, a semi-hollow annular sleeve is processed by a numerical control machine tool and is used as a template for preparing a PDMS annular structure subsequently. The annular sleeve is close to a circular ring structure, but one end of the annular sleeve is solid, so that the annular sleeve is considered as a counterweight on one hand, and the PDMS is prevented from penetrating on the other hand. The main parameter of the sleeve is its outer diameter D, which determines the total area S of the confinement region, where s=pi D ² /4。

4-2) preparation of PDMS ring structures based on semi-hollow annular sleeves. And (3) standing the sleeve in the central area of a 35mm culture dish, and dripping a small amount of deionized water in the central area of the sleeve for temperature measurement of subsequent hot plate heating. And mixing the PDMS prepolymer and the curing agent according to a proper proportion, fully stirring, and placing the mixture in a low-temperature environment in a dark place to remove bubbles. After the bubbles were completely removed, the PDMS mixture was poured into a 35mm dish and left to stand slightly until the surface of the solution was level. And then placing the culture dish on a hot plate, inserting a temperature measuring sensor of the hot plate below the liquid level in the annular sleeve, heating the hot plate to 65 ℃, keeping for 30 minutes, after the PDMS mixed solution is slightly solidified, transferring the culture dish to a constant temperature incubator at 65 ℃ after the sleeve position is fixed, taking out after two hours, sequentially taking out the central annular sleeve, and stripping the PDMS annular structure from the culture dish to obtain the PDMS annular structure with complete structure.

4-3) processing the pourable channel based on a PDMS annular structure. Drilling holes in adjacent 180-degree areas on the side wall of the PDMS annular structure by using a drill gun with a 2.5mm drill bit, flushing with 70% ethanol solution, and drying. And (3) similarly, punching holes at the corresponding positions of the side wall of the 35mm culture dish which is subjected to agarose surface modification and printed with the protein patterns by using a 4mm drill, stopping punching when the holes are nearly punched, puncturing the side wall from inside to outside by using tweezers, and then blowing nitrogen from inside to outside to blow away tiny impurities adhered on the side wall. The method comprises the steps of pressing and fixing a drilled PDMS annular structure to a corresponding position of a 35mm culture dish, then connecting a rubber pipeline with the outer diameter of 2mm into the culture dish, extending the pipeline from outside to inside, stopping slightly extending the inner wall of the annular structure, dripping preheated and dissolved 3% agarose solution into the joint of the pipeline and the inner wall by using a Pasteur pipe, dripping preheated and dissolved 3% agarose solution into the joint of the pipeline and the outer wall of the annular structure by using a Pasteur pipe, placing the 35mm culture dish containing the PDMS annular structure and the 2mm pipeline into the 100mm culture dish, covering the culture dish, standing in a fume hood to reduce the temperature, and fixing the 2mm pipeline after the agarose solution is solidified. If perfusion culture is not added, the outlet of the pipeline extending from the culture dish is clamped by a clip, and the clip is soaked in 70% ethanol for 60 minutes before use and is sterilized by ultraviolet irradiation for the whole night. When perfusion culture is added, paperclips at two ends are taken down, the tail end of a 2mm pipeline at one end is connected with a 1mL syringe through a luer valve, a culture solution containing a specific concentration of medicine is filled in the syringe, the syringe is fixed on a syringe pump, the tail end of the pipeline at the other end is connected with a 5mL syringe containing waste liquid, the syringe is open, the tail end of the pipeline is lower than the liquid level in a culture dish, the tail end of the pipeline is provided with a valve, when liquid is required to be discharged, the valve is opened, the liquid in the culture dish is discharged from the pipeline under the action of gravity, the tail end of the pipeline is adjustable, so that the liquid discharge speed can be adjusted, the structure of the culture dish with the protein pattern printed after the constraint of the obtained PDMS annular structure is shown in figure 8, and the 35mm petri dish contains an Agarose coating, a printed protein pattern and a PDMS annular constraint and a perfusion channel.

The invention adds a constraint to reduce the exposed area of the culture dish substrate prior to step 104, so that less neurons can be inoculated to meet the requirements; and a pipeline is added to realize the perfusion function.

Step 104, primary cortical neuron inoculation is carried out on the culture dish printed with the protein pattern, and an in-vitro modularized neuron network is obtained, which specifically comprises the following steps:

5-1) cortical neurons were purchased from E18-fetuses, in an amount of 1 million, for dry ice transport. Temporary storage is carried out after hands are reached, and the materials are put in a refrigerator at the temperature of minus 80 ℃ for two days and then are put in liquid nitrogen for preservation.

5-2) preparing a primary neuron culture solution. It contained 2% B27,1% glutamax, balance Neurobasal solution.

5-3) 4 35mm dishes with agarose modified and protein pattern printed on the bottom of the dish with PDMS ring structure were prepared in advance. Before inoculation experiments, the freezing tube is taken out of liquid nitrogen, rapidly placed in a 37 ℃ water bath box, and rotated clockwise until a small amount of ice blocks remain in the freezing tube. Taking out from the water bath tank, spraying alcohol for disinfection, and placing in a fume hood. The freezing tube contains about 1mL of freezing solution. The freezing tube was opened and the frozen stock solution was removed to a 15mL centrifuge tube with 5mL of primary nerve culture medium added in advance. At this point the centrifuge tube contained 6mL of neuronal suspension. Taking 1,1.35,1.65,2mL of the suspension into 35mm culture dishes, transferring the culture dishes into an incubator, wherein the culture environment is 37 ℃, the culture environment is 5% carbon dioxide, and the nominal inoculation densities are 653, 882, 1078 and 1307/mm respectively ² . To increase the actual inoculation density, after inoculation, the culture dish was taken out every 30 minutes, and was put into the dish after shaking 10 times in the front-back and left-right directions, respectively. This procedure can be repeated 3-5 times, after which the dishes are left in the incubator for one day, and after which the culture medium is replaced to remove non-adherent neurons. Thereafter, half of the culture medium was replaced every three days.

5-4) cortical neurons self-assemble to form a modular neuronal network. After day 3, neurons began to spread axons on a large scale. After the seventh day, synapses begin to form on a large scale. After the tenth day, the neuron network is basically mature, and can be used for functional tests such as calcium imaging or patch clamp and the like to analyze the influence of the number of neurons in different modules, the density of the neurons and the communication strength of the neurons among the modules on the information transmission of the network, the dynamic characteristics of the network and the like. In combination with perfusion channels, it can also be used to test the interaction between the above variables and different drug stimuli.

The invention has the characteristics and beneficial effects that:

1) The method is based on a digital image processing technology, patterns covering all structural parameter combinations to be researched can be generated and spliced into a single pattern, and a micro printing plate containing all patterns to be researched can be prepared by combining a soft lithography process based on laser direct writing and a surface modification method, so that a modularized protein pattern containing all parameter combinations can be generated by single printing.

2) The method utilizes the micro-fluidic technology to carry out open type constraint on the substrate subjected to surface modification and patterning, and has the function of supporting contact electrophysiological detection on the basis of effectively improving inoculation density and having a pourable channel to support the function of accurate drug testing.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.

The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.

Claims

1. A method of constructing an in vitro modular neuronal network, the method comprising the steps of:

primary cortical neuron inoculation is carried out in the culture dish printed with the protein pattern, and an in-vitro modularized neuron network is obtained;

the method for generating mask patterns covering all structural parameters to be researched by adopting a digital image processing method comprises the following steps:

programming a modularized pattern with adjustable drawing parameters;

adjusting parameters of each modularized pattern with adjustable parameters in the spliced patterns to generate mask patterns covering all structural parameters to be researched;

the modular pattern with adjustable programming drawing parameters specifically comprises:

the upper left corner in the module numerical matrix is ((i-1). Times.2n+round (n/(X) _con +1))×(k-1)-x _width The coordinates of the lower right corner element are ((i-1) ×2n+round (n/(X)) _con +1))×(k-1)+x _width Setting all elements in the (j-1) multiplied by 2n+3n/2) area to 255, and finishing drawing of a transverse connection band between the j-th and j+1th modules of the i-th row; wherein k traverses from 1To X _con ，X _con For the number of transverse connecting bands between the j-th and j+1th blocks of the i-th row, round () is the rounding operator, x _width A bandwidth of a transverse connection band between the jth and the (j+1) th modules of the ith row;

the upper left corner is ((j-1) ×2n+round (n/(Y) _con +1))×(k'-1)-y _width The coordinates of the lower right corner element are ((j-1) ×2n+round (n/(Y)) _con +1))×(k'-1)+y _width 2, setting all elements in the (i-1) multiplied by 2n+3n/2) area as 255, and finishing drawing of longitudinal connection bands between the ith and the (i+1) modules of the jth column; wherein k' traverses from 1 to Y _con ，Y _con The number of longitudinal pass bands between the ith and (i+1) th modules in the jth column, y _width The bandwidth of the longitudinal passband between the ith and the (i+1) th module of the jth column;

splicing a plurality of modularized patterns with adjustable parameters to obtain spliced patterns, wherein the splicing method specifically comprises the following steps of:

2. The method of constructing an in vitro modular neuronal network according to claim 1, wherein the adjustable parameters in the parameter adjustable modular pattern comprise: the side length of the modules, the center distance of the modules, the number of communication bands between the modules and the width of the communication bands are used for adjusting the number of neurons, and the center distance of the modules, the number of the communication bands between the modules and the width of the communication bands are used for adjusting the communication intensity between the neurons.

3. The method for constructing an in-vitro modularized neuron network according to claim 1, wherein the soft lithography process using laser direct writing is used for preparing a PDMS micro-printing plate according to the mask pattern, and specifically comprises:

developing and etching the chromium plate after masking to obtain a die;

4. The method for constructing an in vitro modularized neuron network according to claim 1, wherein the protein pattern is generated according to the PDMS microprint in the surface-modified culture dish, and the culture dish printed with the protein pattern is obtained, specifically comprising:

5. The method of claim 1, wherein primary cortical neuron seeding is performed in the protein pattern-printed culture dish to obtain an in vitro modular neuronal network, further comprising:

placing a semi-hollow annular sleeve in the central region of a 35mm dish;